Human Umbilical Cord Wharton Jelly Cells Treatment Prevents Osteoporosis Induced by D-Galactose

Methods Sixteen male mice were randomly divided into 4 groups: control (ordinary feeding), D-gal (D-galactose) group, D-gal + MSC (human umbilical cord Wharton jelly cells), and D-gal + MSC-TNFα groups. Except for the control group (fed with same amount of saline solution), other mice received gastric feeding of 250 mg/kg D-galactose every day for 8 weeks. TNFα (10 ng/mL for 24 h) cocultured or noncocultured HUCWJCs (5 × 105) were suspended in 0.1 ml of sterile PBS and injected into tail veins every other week in D-gal + MSC-TNFα and D-gal + MSC groups, respectively, and only 0.1 ml of sterile PBS for control and D-gal groups. The bone mass was detected by qPCR, ELISA, microcomputed tomography (μCT), and hematoxylin-eosin staining. Proliferation, apoptosis, and differentiation of periosteal-derived osteoblasts (POB) were assessed. Transwell assay and scratch healing were performed to detect POB migration and invasion ability. The effect of HUCWJCs on POB signaling pathway expression was evaluated by immunoblotting. Results The malondialdehyde (MDA) in serum was higher and superoxide dismutase (SOD) was lower in the D-gal group compared to the other groups (p < 0.05). Mice in D-gal group showed significantly decreased bone mass when compared to the control group, while HUCWJCs treatment partially rescued the phenotype, as demonstrated by μCT and histology (p < 0.05). Mechanically, HUCWJCs treatment partially promoted proliferation and migration and decreased apoptosis of POB induced by oxidative stress via activating the mitogen-activated protein kinase (MAPK) signaling pathway. Conclusion HUCWJCs can prevent the progression of osteoporosis by inhibiting oxidative stress, which may act by regulating osteoblasts fate through the MAPK signaling pathway.

Introduction
Osteoporosis is a common disease characterized by decreased bone density and increased risk of fracture that affects hundreds of millions of people worldwide [1]. Many studies suggested that the increased oxidative stress level is closely related to the occurrence of osteoporosis [2][3][4]. Oxidative stress has a key role in transmitting intracellular signals that regulate proliferation, differentiation, repair process, and inflammation of cells [5]. It was suggested that oxidative stress inhibits bone formation [6]. In addition, decreased glutathione levels and defensive antioxidant capacity have been found in elderly patients with osteoporosis and secondary osteoporosis caused by chronic intestinal disease (IBD) [7,8]. e role of D-galactose in inducing oxidative stress has been widely used to establish animal model of premature aging and the aging process induced by D-galactose is like the natural aging process [9][10][11]. Human umbilical cord Wharton jelly cells (HUCWJCs) have been used to treat various inflammatory diseases due to their excellent anti-inflammatory properties. Previous studies have shown that the decrease of resident mesenchymal stem cells is closely related to the occurrence of osteoporosis. Exosomes from human adipose-derived mesenchymal stem cells have been often used for bone tissue engineering and repair of bone defects [12]. ese exosomes ameliorate inflammation and promote alkaline phosphatase (ALP) activity, calcium deposition, osteocalcin (OCN) secretion, and proliferation and inhibit apoptosis in osteoblasts [13]. Ge et al. suggested that these exosomes are sufficient to reverse the symptoms of osteoporosis in mice [14]. So far, there is no literature to report whether the D-galactose-induced osteoporosis mouse model can be treated by HUCWJCs. We hypothesized HUCWJCs could be a mediator in osteoporosis therapy and pro-inflammatory factors (TNFα) may inhibit its anti-inflammatory effects. To address the question, we characterized oxidative stress in mice, after which the bone mass of lower limbs was quantified and the signaling pathway expression was explored.

Animals.
Since estrogen can prevent the development of osteoporosis and its anti-inflammatory effect interferes with the establishment of D-galactose induced osteoporosis model, male mice are more suitable for this study [15,16]. Accordingly, sixteen wild-type male C57BL/6J mice were purchased from Shanghai SLAC Laboratory Animal Company Limited. All the animals were housed in an environment with a temperature of 22 ± 1°C, a relative humidity of 50 ± 1%, and a light/dark cycle of 12/12 hr and were given water and food ad libitum. e dose of D-galactose inducing oxidative senescence in mice (200 mg/kg/d for eight weeks) was taken for reference [17,18]. Human umbilical cord Wharton jelly cells (HUCWJCs) can inhibit the occurrence of osteoporosis by inhibiting expression of TNFα and also exhibit good antioxidant effect by inhibiting TNFα related signaling pathways [19]. However, it was also suggested that TNFα promotes osteogenic differentiation of HUCWJCs [20]. To explore whether TNFα inhibits the anti-inflammatory effect of HUCWJCs and intervenes the effect of HUCWJCs on osteoporosis, in our study, mice were randomly divided into 4 groups: control (ordinary feeding), D-gal (D-galactose) group, D-gal + MSC (human umbilical cord Wharton jelly cells), and D-gal + MSC-TNFα groups. All mice were fed conventional diet. At 6 months of age, except for control group, all mice received gastric feeding of 250 mg/kg D-galactose every day for 8 weeks. At the same time, TNFα (10 ng/ mL for 24 h) cocultured or noncocultured HUCWJCs (5 × 10 5 ) were suspended in 0.1 ml of sterile PBS and injected into tail veins every other week in D-gal + MSC-TNFα and D-gal + MSC groups, respectively, and only 0.1 ml of sterile PBS injection for control and D-gal groups. At 8 months of age, blood was collected and analyzed by ELISA (Jiangsu Yutong Biological Technology Company). Mice were then euthanized using pentobarbital (100 mg/kg, IP injection), and the lower extremities of the sacrificed mice were dissected and immediately put in 4% paraformaldehyde for 24 h and then transferred to phosphate-buffered saline. e experiments were performed in accordance with the requirements of the local ethical committee. Arteries and vessels were removed from the umbilical cord. Wharton's jelly was split into pieces and cultured in DMEM/F12 supplemented with 1% nonessential amino acids, two mmol/L glutamine, 10% heat-inactivated FBS, 100 U/mL penicillin, and 100 g/L streptomycin in an incubator at 37°C with 5% CO 2 . One week later, a cell clone of HUCWJCs could be observed around Wharton's jelly slices. HUCWJCs were cocultured with TNFα (10 ng/mL R&D Systems) for 24 h before infusing into a caudal vein, which was referred to relevant literature [21,22]. Periostealderived osteoblasts (POB) were provided by arthrosis colleagues. POB pretreated with or without H 2 O 2 (400 uM) for 3 h were ROS and control group, respectively, while POB pretreated with H 2 O 2 and followed by culturing with HUCWJCs or HUCWJCs-TNFα for 48 h were ROS + MSC group and ROS + MSC-TNFα group, respectively. is grouping method is applicable to the subsequent grouping of experimental steps, including the EDU, TUNEL, mineralization assay, migration, invasion, and signaling pathway expression examination.

Micro-CT.
e right lower extremities were harvested from the mice following euthanasia. Samples were fixed in 4% paraformaldehyde for 24 h and then scanned using a micro-CT system (MicroCT 40 and vivaCT; ScancoMedical, Bassersdorf, Switzerland). Global thresholding procedure was used to separate the calcified tissues from soft tissues. e distal epiphyseal of femur was scanned at 8 μm resolution to analyze trabecular bone mass. e following parameters were determined for the trabecular bone at the distal femur: bone mineral density (BMD), relative trabecular bone volume (BV/TV), trabecular spacing (Tb. Sp), trabecular number (Tb. N), and trabecular surface area/bone volume (Tb.BS/BV). ree individuals, all of whom were blinded to the grouping, performed the radiographic analysis.

Histology and Mineralization Assay.
After μCT test, the right lower extremities were treated with 0.25 M ethylenediaminetetraacetic acid (EDTA) for decalcification for 3 weeks. e samples were then embedded in paraffin, and 6 μm sections were prepared using a standard microtome (RM2255, Leica). Sections were stained with hematoxylineosin (H&E) (SD0146, Shanghai Si Ding Biological Technology Company). For H&E staining, trabecular bone area/ total area was calculated with Image-Pro Plus V6.0.0. For mineralization assay, four groups of cells were treated under different conditions (as described above), then followed by culturing with osteogenic differentiation medium including αMEM supplemented with 10% fetal bovine serum (FBS), 1% streptomycin (100 mg/mL), penicillin (100 u/mL), 100 nM dexamethasone (Sigma), and 50ug/mL ascorbic acid 2 International Journal of Clinical Practice (Sigma) as well as 10 mM β-glycerophosphate sodium (Sigma). Consequently, cells were subjected to Alizarin Red S staining at day 14 (60504ES25, YEASEN, China) to identify calcium deposits in the cell layers of osteoblasts. Images were acquired using a microscope (Olympus, Tokyo, Japan). For quantitative analysis, cells were destained with ethyl pyridine chloride for 30 min at room temperature and transferred to a 96-well plate to measure the absorbance at 550 nm using a microplate reader.

Real-Time RT-PCR Analysis.
e left lower extremities from 8-month-old mice were dissected. e samples were subsequently placed in Trizol (15596026, ermo Fisher Scientific, USA). Total RNA was then extracted according to the manufacturer's instructions, and cDNA was synthesized from 1 μg of total RNA. qPCR was performed with SYBR Green PCR Master Mix (Q711-02, Vazyme, USA).

TUNEL Assay.
Terminal deoxynucleotidyl transferasemediated dUTP-biotin Nick End Labeling (TUNEL, Beyotime, Beijing, China) was used to visualize cell apoptosis. POB were suspended in a serum-free medium and plated in the upper chamber at a density of 4 × 10 4 cells/dish and the four groups of POB were treated under different conditions. Apoptotic cells were then counted under 5 visual fields with FLUOVIEW software. TUNEL staining was performed by three researchers who were blinded to the grouping.

Transwell Assay and Scratch-Wound
Assay. For analysis of invasion capability, POB were suspended in 250 μL serum-free DMEM/F12 and added into the upper chamber of a Transwell plate (Corning, New York, NY, USA) with an 8 μm pore size polycarbonate filter. After complete culture medium with 10%FBS with or without H 2 O 2 (400 uM for 3 h), the medium was changed into a 600 μL complete culture medium with 10% FBS + HUCWJCs/HUWJCs-TNFα in the lower chamber. After 48 hours, cells in the lower chamber were stained by crystal violet, and a cotton swab was used to remove cells on the upper surface of the filter and observed under an inverted microscope. For scratch-wound assay, after the cell reached 90% confluence, a line was drawn using a marker on the bottom of the dish, and then a sterile 1,000 μl pipet tip was used to scratch three separate wounds through the cells, moving perpendicular to the line. e cells were gently rinsed twice with PBS to remove floating cells and incubated in 5 ml of medium containing 10% FBS in 37°C, 5% CO2/95% air environment. Images of the scratches were taken by using an inverted microscope after 0.12 and 36 h of incubation and quantified by calculating the wound healing area of cells in the scratched area.

Statistical
Analysis. Statistical analysis of the data was performed by t-tests or one-way analysis of variance (ANOVA). All data are presented as means ± SD. p < 0.05 was considered as statistical significance. Each experiment was performed in triplicate.

HUCWJCs Partially Rescue the Osteoporosis Phenotype
Induced by Oxidative Stress. To investigate the effect of HUCWJCs on osteoporosis in mice, we established the osteoporosis model by D-galactose induction. After 8 weeks of feeding with 250 mg/kg D-galactose and HUCWJCs treatment, malondialdehyde (MDA) in serum in D-gal group (1.768 ± 0.185umol/L) and in D-gal + MSC group (1.220 ± 0.091 umol/L) was both higher than that in control group (0.442 ± 0.261 umol/L) (all p < 0.05). Moreover, superoxide dismutase (SOD) in D-gal group (331.033 ± 4.535 umol/L) was lower than that in control group (359.887 ± 9.103 umol/L) (p < 0.05), while there was no significant statistical difference between D-gal + MSC group (351.620 ± 5.021 umol/L) and control group (p > 0.05), which means that the inflammation level of mice was increased and the antioxidant capacity was downregulated after D-galactose induction (Figure 1(a)). Alkaline phosphatase (ALP) in serum in D-gal group (2.398 ± 0.116 mmol/L) and D-gal + MSC group (2.595 ± 0.048 mmol/L) was also lower compared to the control group (2.735 ± 0.435 mmol/L) (p < 0.05); In addition, parameter of ALP in D-gal + MSC group was significantly higher than the D-gal group (p < 0.05). ere is no statistical significance between D-gal + MSC and D-gal + MSC-TNFα groups (Figure 1(b)). μCT showed the lowest bone mass at the distal femur in the D-gal group after treatment for 8 weeks.  (Figures 1(c) and 1(d)). Taking the control group as a reference, H&E stains showed the trabecular bone area/ total area at distal femur in D-gal group was 0.613 times that of control group (p < 0.05), and the ratios in D-gal-MSC (0.944 times of control group) and D-gal-MSC-TNFα groups (0.810 times of control group) were higher to that of D-gal group (p < 0.05), while there is no statistical difference between the . All data is reported as the mean ± SD. Statistical significance was determined by one-way ANOVA. * p < 0.05 , * * p < 0.01, * * * p < 0.001; ns � not statistically significant. (Figure 1(e)).

HUCWJCs Treatment Rescues Osteoblasts Differentiation, Proliferation, and Apoptosis Induced by Oxidative Stress
In Vitro. Identification of stem cell pluripotency of HUCWJCs toward osteoblasts, adipocytes, and chondrocytes was confirmed in vitro (Figure 2(a)). e expression of osteogenic mRNA (ALP, OCN, and OPN) was significantly decreased in the ROS group compared to the ROS + MSC and control groups (p < 0.05, Figure 2(b)). Besides, the mRNA expression levels of OCN and OPN were significantly lower in ROS + MSC-TNFα group when comparing to the ROS + MSC group with statistical difference (p < 0.05). POB were then subjected to Alizarin Red S staining at day 14 (60504ES25, YEASEN, China) to identify calcium deposits in the cell layers, followed by quantification. e ROS group showed less osteogenic ability compared to the control group, while the osteogenic ability of ROS + MSC group and ROS + MSC-TNFα group was significantly stronger than that of ROS group, and there was no significant difference between ROS + MSC group and ROS + MSC-TNFα group (Figure 2(b)). To gain insights into the cellular mechanism of osteogenesis defects caused by oxidative stress, EDU & TUNEL staining were performed to detect proliferation and apoptosis of POB. Interestingly, the results indicated that the proliferation of POB decreased (p < 0.05), and the level of apoptosis was upregulated in the ROS group compared to the control group (p < 0.05). HUCWJCs treatment promoted proliferation and inhibited POB apoptosis (p < 0.05). Meanwhile, there is no statistical difference of proliferation and apoptosis between the ROS + MSC and ROS + MSC-TNFα groups (p > 0.05, Figure 2(c) and 2(d)).

HUCWJCs Treatment Enhances the Invasion and Migration Ability Induced by Oxidative Stress.
Next, we investigate the effect of HUCWJCs on the invasion and migration ability of POB, which were precultured with or without H 2 O 2 (400 uM) for 3 h, followed by coculturing with HUCWJCs or HUCWJCs-TNFα for different periods of time (0,12,36 h). e wound healing percent in the scratched area in ROS group (0.959 ± 0.275%) is lower than that in ROS + MSC group (10.369 ± 1.118%) and control group (6.454 ± 0.695%) at 36 h (Figure 3(b), p < 0.05). ere was no statistical difference between control group and ROS + MSC group at 36 h. For invasion assay, with control group as the reference, cells stained by crystal violet were 16.51 ± 2.22% in ROS group. And crystal violet stain area ratio in ROS-MSC group (28.62 ± 2.92%) was higher than that in ROS group (16.51 ± 2.22%) and that in ROS + MSC-TNFα group (21.74 ± 3.10%) (Figure 3(a), all p < 0.05).
is further suggested that HUCWJCs treatment enhances the invasion and migration ability induced by oxidative stress.

HUCWJCs Activate the Mitogen-Activated Protein Kinase Signaling Pathway Expression of POB.
To investigate the signal transduction system for extracellular signals to intracellular reactions in different groups, protein expression of the mitogen-activated protein kinase pathway was detected. e expression of phosphorylated ERK1/2 to total ERK1/2 in ROS group was 0.388 times that in control group and was 0.504 times that in ROS + MSC group (p < 0.05). e expression of phosphorylated p38 to total p38 in ROS group was 0.335 times that in control group and was 0.211 times that in the ROS + MSC group (p < 0.05). And ROS + MSC-TNFα group had lower expression of phosphorylated ERK1/2 and phosphorylated p38 than that in ROS + MSC group (p < 0.05). e data was quantified by ImageJ ( Figure 4).

Discussion
D-galactose has been widely used to establish aging model in mice. According to Qian, chronic D-galactose exposure decreases the activity of catalase and increases the accumulation of lipid peroxidation, advanced glycation end products (AGEs), and malondialdehyde (MDA) [18,23]. D-galactose has been shown to promote senescence of neural stem cells and neurons through oxidative stress and proapoptotic forms [24]. In our study, D-galactose was used to induce osteoporosis in mice, MDA in serum of D-gal group was significantly higher than that in D-gal + MSC and control groups, and mice in D-gal group showed significantly decreased bone mass when compared to the control group, indicating the oxidative stress-induced osteoporosis model in mice is successful. HUCWJCs have unique and valuable inherent characteristics: first, they can be easily obtained without any risk to the donor and with no ethical controversy; second, HUCWJCs possess low immunogenicity and good antioxidant ability [25][26][27], which means low concentration of reactive oxygen species and a high concentration of glutathione (key antioxidant). To further determine how oxidative stress and HUCWJCs treatment affect osteogenesis, the trabecular bone mass at the distal femur was assessed in four groups in our study. Parameters of BMD, Tb. N, and BV/TV at distal femur in D-gal-MSC group were significantly higher than those in D-gal group, which suggests that HUCWJCs prevent the progression of osteoporosis. HUCWJCs-derived extracellular vesicles serve as a critical regulator of bone metabolism and represent a potential agent for prevention and treatment of osteoporosis [28]. Zhang et al. reported coculture of osteoblasts and HUCWJCs promoted the proliferation and osteogenic ability of osteoblasts [29,30]. HUCWJCs-exosomes also promote cell proliferation and protect against oxidative stress-induced cell apoptosis in vitro by activation of ERK1/2 and p38 [31]. We confirmed that HUCWJCs treatment partially promoted proliferation and migration and decreased apoptosis of POB induced by oxidative stress via activating the mitogen-activated protein kinase pathway in vitro.
Epidemiological studies showed that patients under oxidative stress are more prone to osteoporosis [32].  International Journal of Clinical Practice Oxidative stress has been strongly associated with osteoporosis [33,34]. We identified osteoporosis progresses in the D-gal group. Yet, no study has reported whether the D-galactose-induced osteoporosis mouse model can be treated by HUCWJCs. So, what is the relationship between the ability of HUCWJCs to inhibit oxidative stress and osteoporosis? D-galactose induces oxidative stress via elevated levels of oxide in the blood [35]. is further triggers an inflammatory response in the plasma by increasing proinflammatory cytokines (including TNFα) and decreasing anti-inflammatory cytokine [36]. e imbalance between pro-and anti-inflammatory cytokine has been considered a causative factor of diminished bone mineral density in osteoporosis [37]. Also, proliferation and differentiation of osteoblasts could be induced by stimulating ERK, JNK, and p38 [38,39], while oxidative stress could inhibit the MAPK signaling pathway and induce apoptosis of cells [40,41]. As reported previously, MSC-derived exosomes inhibit osteoporosis by promoting the proliferation of osteoblasts via the MAPK pathway [42]. erefore, the suppression of inflammation by HUCWJCs helps prevent the development of osteoporosis by balancing inflammatory factors and maintaining the activity of osteoblast MAPK signaling pathway. Also in our study, we found the proliferation and migration abilities of POB decreased in the ROS group when compared to the other groups, while HUCWJCs treatment partially promoted proliferation and migration and decreased apoptosis of POB induced by oxidative stress by activating the MAPK signaling pathway.
In addition to the antioxidant capacity of HUCWJCs, we believe that there are two main reasons explaining why HUCWJCs inhibit the process of osteoporosis. Firstly, altered differentiation potential of MSCs that favor adipocytes rather than osteoblasts is one main cause for osteoporosis; therefore, a treatment strategy aimed at augmenting endogenous MSCs could be a potential method for osteoporosis therapy [43]. Secondly, in vivo trials suggested that MSC-exosome promotes angiogenesis, which in turn prevents the process of osteoporosis [44]. In addition, to investigate whether HUCWJCs affect the progression of osteoporosis through inflammation, TNFα, one of the major inflammatory factors, was included in our study. We hypothesized that coculture of TNFα and HUCWJCs would inhibit HUCWJCs' anti-inflammatory and antiosteoporosis effects. However, in the whole experimental process, compared with the D-gal + MSC group, the D-gal-MSC-TNFα group showed a certain degree of bone mass reduction in μCT and histology, but there was no statistical difference. Only the expression of osteogenesis related mRNA and the changes of invasion and migration in vitro showed statistical difference. is was not exactly consistent with our expectation, and we believed that there were several probable causes for this result. All data is reported as the mean ± SD. Statistical significance was determined by one-way ANOVA. * p < 0.05, * * p < 0.01, * * * p < 0.001; ns � not statistically significant.

International Journal of Clinical Practice
HUCWJCs, which may be related to the above reasons and needs to be further studied.

Conclusion
HUCWJCs can prevent the progression of osteoporosis by inhibiting oxidative stress, which may act by regulating osteoblasts fate through the MAPK pathway.
Data Availability e data that support the findings are available from the corresponding author (Desheng Wu) upon reasonable request.

Ethical Approval
All animal studies (including the mice euthanasia procedure) were done in compliance with Shanghai East Hospital institutional animal care regulations and guidelines and conducted according to the AAALAC and the IACUC guidelines (Animal Research: Reporting of In Vivo Experiments).

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this article.


Throughput screening of Bacillus subtilis strains that abundantly secrete surfactin in vitro identifies effective probiotic candidates

Since the prohibition of antibiotics as animal growth promoters, demand for effective probiotic strains has steadily increased. The goal is to maintain productivity and mitigate environmental concerns in the livestock industry. There are many probiotic animal-diet supplements available, over 2,000 products in the Republic of Korea alone, with little explanation about the desirable properties of each probiotic strain. The purpose of this study was to describe the underlying logic and methods used to select two novel strains of probiotic candidates. To economically screen these candidates, the abundance of surfactin secreted was used as an in vitro marker. We used a modified oil-misting method to screen ~2,000 spore-forming bacteria for novel strains of Bacillus subtilis. Of these, 18 strains were initially selected based on the semiquantitative criterion that they secreted more surfactin than B. subtilis ATCC21322 on Luria-Berani (LB) agar plates. The whole genome sequence was determined for two of the 18 strains to verify their identity. A phylogeny of 1,162 orthologous genes, genome contents, and genome organization confirmed them as novel strains. The surfactin profiles produced by these two strains consisted of at least four isoforms similar to standard surfactin and enhanced cellulase activities up to 50%. Four fractionated individual isoforms of surfactin suppressed inflammation induced by lipopolysaccharides. The half-maximal inhibitory concentration (IC50) was about 20 μM for each isoform. Both selected strains were susceptible to seven important antibiotics. Our results implied that an abundant secretion of surfactin was a useful biomarker in vitro and could be utilized for mining probiotic candidates through high-throughput screening of environmental samples.

Introduction
Multidrug-resistant bacterial strains and environmental pollution that originates from intensive animal farming could become serious social and medical problems. Such concerns led the industry from a reduction in the use of antibiotics to their total ban as animal growth promoters, including a massive reduction as therapeutics [1]. Beneficial microorganisms known as probiotics have emerged as growth promoters and viable alternatives to antibiotics and harmful chemicals. Spore-forming bacteria are potentially promising probiotics [2]. Unlike vegetative cells that are usually killed by gastric and bile fluids, bacterial spores survive the harsh conditions of the gastrointestinal tract and reach the large intestine of farm animals and humans [3,4]. They germinate in the digestive tract and newly germinated vegetative cells persist without active proliferation [4][5][6]. Vegetative cells would remain metabolically active, secreting bacteriocins important for competing with pathogenic bacteria in the digestive tract [7]. Important properties of probiotic strains for livestock are directly associated with enhancing productivity and animal welfare, and possibly the reduction of noxious gas emission resulting from a decreased production of methane, ammonia, hydrogen sulfide, and carbon dioxide [8]. Several bacterial genera have been developed as probiotics, including Lactobacillus, Bifidobacterium, and Bacillus. Interest in Bacillus spp. as new probiotics has been steadily increasing, partly because they form spores. Whole-genome sequences for 11,613 Bacillus strains were listed in NCBI on May 2, 2022. The list included 1,016 strains of B. subtilis, 427 strains of B. licheniformis, 263 strains of B. amyloliquefaeciens, and 855 strains of B. velezensis. Some of these strains have been developed as probiotics. Nonetheless, the need for probiotics is growing, and screening novel strains with high efficacy is still necessary.
Functional testing in vivo to evaluate their probiotic effect on the animal host is a long, expensive effort compared to in vitro testing. It is therefore beneficial to select a small number of promising candidates by massive in vitro screening before performing in vivo tests. Characteristics associated with in vivo function, and that can be evaluated by in vitro tests, however, are poorly defined. We decided for several reasons to test whether the abundant secretion of surfactants in vitro was a potential biomarker for in vivo functionality. First, biosurfactants secreted by several Bacillus species have antimicrobial properties comparable to conventional chemical drugs [7]. They include lipopeptides like surfactin, fengisin, and iturin. We hypothesized that the abundant secretion of antibacterial surfactants was a desirable feature for suppressing infectious microbes and preventing diseases in animals. It may have an effect similar to antibiotics. Although secretion of any antimicrobial peptide would prevent the colonization of the intestine by pathogenic bacteria, we preferred to screen surfactant-secreting strains because surfactin also has anti-viral, anti-fungal, and anti-mycoplasma activities [9].
For high-throughput screening, we adopted the oil-misting method of Burch and colleagues [10]. This method does not require a liquid culture step and the results are straightforward and fast to analyze. In this study, we discovered two novel B. subtilis strains that abundantly secrete biosurfactants in vitro. We then examined the properties of surfactin that pertain to probiotic functions, and discuss possible reasons for considering them as potent probiotic candidates.

Screening spore-forming and surfactant-producing bacteria
To discover surfactant-producing bacteria among heat-tolerant microorganisms, a large number of microbial colonies were screened by a modified airbrush method [10]. First, 0.5 g of a dried environmental sample, such as soil or animal feces, was ground by mortar and pestle. The powder was put in a tube suspended in 50 mL of distilled water preheated to 80˚C, followed by incubation in an 80˚C water bath for 5 min. The tube was then centrifuged at 3,000 g for 10 min to precipitate the heavier substances. The slightly opaque layer of supernatant formed was spread on LB plates and incubated at 37˚C overnight. Next, well-separated bacterial colonies from the LB plates were spotted onto fresh LB agar plates using sterile yellow pipette tips and incubated overnight. Bacillus subtilis strain ATCC21322 was also inoculated onto each plate as a positive control. An airbrush, Beetle Bug (Yamato comp., Seoul, ROK), was used to apply a fine mist of canola oil onto the plates at an air pressure of 2 bars. The distance between the air brush and plate was set to deposit a fine mist of oil droplets, making biosurfactant halos visible to the eye without special equipment. The size and transparency of the halo correlated with the amount of secreted surfactant [10]. Halo quality was visually assessed by clarity, transparency, and evenness of the oil layer in the clear zone, using three grades: 1) opaque with numerous small oil drops, 2) medium clear with a small number of oil drops, and 3) clear with no visible oil drops. Halo size was measured as the diameter of the clear zone from the leading edge, through the center to the other-side edge.

Genome sequencing, assembly, initial annotation
Genome sequences of two B. subtilis strains, ps4060 and ps4100, were determined and assembled at DNA-Link (DNA-Link, Seoul, ROK) using the PacBio platform (Pacific BioScience, Menlo Park, CA) following manufacturer's instruction. We used 5 μg of each sample to construct a SMRTbell library with the SMRTbell™ Template Prep Kit 1.0 (PN 100-259-100). A sequencing primer was annealed to the SMRTbell template over a 20-kb genomic DNA fragment to construct a sequencing library [11]. The library was sequenced using 1 SMRT cell with MagBead OneCellPerWell v1 Protocol for 240 minutes using the PacBio RS II (Pacific Biosciences) sequencing platform. Over 64,000 long reads and 570 million base pairs were identified for each strain. After filtering subreads, the average sequence coverage of the genome was over 110 times. De novo assembly was conducted using the hierarchical genome assembly process (HGAP, Version 2.3) workflow. This process included consensus polishing with Quiver [12]. Since bacterial genomes and plasmids are typically circular, we checked the form of each contig using MUMmer 3.5 [13] and trimmed one of the self-similar ends for manual genome closure. The genome sequences were deposited in GenBank with accession numbers CP081458 and CP076445 for ps4060 and ps4100, respectively. The strains were also deposited in the Korea Microbe Collection Center, Korean Agricultural Culture Collection (KACC, http://genebank.rda.go.kr/), National Institute of Agricultural Science International Depository Authority (https://www.acm-mrc.asia/M/kctc.html), with accession numbers KACC81161BP and KACC81159BP respectively for ps4060 and ps4100.

Gene phylogenies and comparison of bacterial genomes
The genomes of ps4100 and ps4060 each contained ten rRNA operons. The ten 16S rRNA sequences were mined from each genome and examined: only four of them had unique sequences. Other Bacillus species also had multiple copies of rRNA operons in their genomes, from which only unique sequences were subsequently used to construct gene-phylogenies. Accordingly, one copy among identical sequences was used for phylogenetic tree construction. Ribosomal RNA generally reflects organismal phylogeny; however, this gene is also transferable among closely related species [14,15]. In addition, there were insufficient phylogenetic signals for subspecies classification. To minimize rare but possible gene transfer effects on organismal phylogeny and to acquire subspecies-level resolution, additional phylogenies were constructed with single-copy genes shared among B. subtilis strains. Initially, 430 wholegenome sequences were downloaded from NCBI. From these we selected 176 taxa, each with a genome of less than four contigs. The genomes of ps4060 and ps4100 were included in subsequent analyses. Each genome was annotated by the NCBI Prokaryotic genome annotation pipeline (Version 6.1 March 2022). Candidates with common genes were clustered using options of: 70% sequence identity, a 0.9 length difference cutoff, 0.9 alignment coverage, and a 90% length difference imbedded in CD-HIT [16]. After selecting 1,162 clusters, individual gene sequences were concatenated using Python script, followed by multiple sequence alignment using UGENE or MAFFT v4.763 [17,18]. Gaps were removed before phylogenetic analysis using the maximum likelihood method in MEGA [19]. Further, a phylogeny with 13 taxa was constructed with those representing major groups in the B. subtilis phylogeny and Bootstrap analysis was also performed. For visual comparisons of whole genome structure, global alignment of bacterial genomes between most-closely related strains was performed with Mauve [20]. Mauve aligns multiple genome sequences and is especially instrumental for showing a large-scale inversion and genome rearrangement. Options used are 'align with progressi-veMuave, default seed weight, determine LCBs, default mean LCB weight, full alignment, iterative refinement, and sum-of-pairs LCB scoring'.

Production and quantification of surfactin
For surfactin production, we inoculated a 250-mL flask containing 50 mL of surfactin production medium with a loopful of the bacteria. The medium contained 40 g/L glucose, 5 g/L yeast extract, 10 g/L peptone, 3.27 g/L K 2 HPO 4 �3H 2 O, 1.5 g/L NaCl, 0.5 g/L Na 2 CO 3 , 1 g/L MgSO 4 , and 0.91 g/L FeSO 4 �7H 2 O. This composition was determined based on our fermentation experience and three publications [21][22][23]. The flask was agitated by shaking at 150 rpm and 30˚C for 70 h. The culture was transferred to 50-mL centrifuge bottles, centrifuged at 10,000 g, and a cell-free broth recovered by removing the cells and cell debris. The broth was acidified to pH 2-4 by adding 1 M HCl and incubated at 4˚C overnight. A second centrifugation at 13,000 g for 20 min was used to harvest the brown precipitants. The precipitants were stored at 4˚C or -20˚C until use, or directly dissolved in 95% methanol for downstream quantification. Surfactin concentration was determined by reverse phase HPLC equipped with a C18 column (column diameter 5 μm) at 30˚C [24]. The samples were filtered through a 0.45-μm membrane (Corning, Berlin, Germany) before analysis. A mixture of trifluoroacetic acid (20% v/v) and acetonitrile was used as the mobile phase. The flow rate was 1.0 mL/min. Quantitative analysis of each sample was conducted at least twice under identical conditions. Surfactin purchased from Sigma-Aldrich (St. Louis, MO, USA) was used as the standard.

Effect of surfactin on cellulase activity
Surfactin was completely dried and dissolved in methanol (10 mg/mL). The resulting solution was subsequently diluted with deionized water or an appropriate buffer as described for each experiment. Surfactin solution was added to the reaction mixture and cellulase activity measured [25]. The cellulose substrate was dissolved in a large volume of 50 mM acetate buffer (pH 5.0). An addition of deionized water without buffer, or a small amount of 1 M Tris-HCl buffer (pH 8.0) to make a 3 mM concentration, did not affect the pH of the reaction mixture. Spectrophotometric Stop Rate Determination was performed following the protocol of Enzymatic Assay of Cellulase (Sigma-Aldrich, St. Louis, MO, USA). The cellulase solution, 1 unit/ mL in cold deionized water, was prepared immediately before the enzymatic assays. Glucose generated by the enzyme reaction was calculated by the increase in ΔΔA340 nm; (A340 nm Test final-A340 nm test initial)-(A340 nm blank final-A340 nm blank initial). We used ΔΔ to compensate for the possible irregularity of background absorbance in each well. Relative amounts of glucose were calculated by (ΔΔA340 nm of reaction with surfactin-ΔΔA340 nm of reaction without surfactin) / ΔΔA340 nm of reaction without surfactin � 100.

Analysis of nitric oxide production in macrophage cells
We fractionated four major isoforms of surfactin a, b, c, and d (S1 Fig in S1 File) from the total surfactin dissolved in 99.99% methanol by a preparative HPLC system (Agilent Technology, Santa Clara, CA, USA). The fractionated isoforms were completely dried in a SpeedVac vacuum drier (Eppendorf, Hamburg, Germany), and then dissolved in dimethyl sulfoxide (DMSO), which was effective regardless of pH [26]. We used DMSO alone in a negative control experiment. We tested the effect of each isoform on nitric oxide (NO) production as an indicator of inflammatory responses in mouse macrophage RAW 264.7 cells [27,28]. The RAW 264.7 cells were cultured at 5 × 10 4 cells/well in a 96-well microplate in 200 μL of Dulbecco's Modified Eagles Medium (DMEM). The medium contained 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 μg/mL streptomycin. To induce an inflammatory response, we added 0.5 μg/mL lipopolysaccharide (LPS) to each well pretreated for 30 min with different amounts of a surfactin isoform. After the cells were cultured at 37˚C for 24 h, they were centrifuged to separate supernatant and precipitant. The supernatant from the cell culture was recovered after centrifugation and the concentration of NO measured by the NO Plus Detection Kit (iNtRON Biotechnology, Seongnam, Republic of Korea) using the Griess reagent system following the manufacturer's protocol [29]. Absorbance was measured at 540 nm with a Spectra Max 190 microplate reader, (Molecular Devices, San Jose, USA) by converting the NO concentration in the supernatant using a standard curve for each concentration of sodium nitrite. The half-maximal inhibitor concentration (IC 50 ) was obtained using GraphPad Prism (GraphPad Software, 8.4.3, San Diego, CA, USA).

Measurement of iNOS protein
The RAW 264.7 cells were treated with 0.5 μg/mL LPS to induce an inflammatory response. Surfactin isoforms A, B, C, and D were individually added to the cells at 10 and 25 μM concentrations, followed by 24 h at 37˚C in the presence of 5% CO 2 . We used a Western blot to examine iNOS expression after collecting the cells from each treatment and washing them twice with PBS. Cell lysis buffer (10 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X, 1 mM PMSF, 1 μg/mL aprotinin, 1 μg/mL leupeptin, 1 mM DTT) was added and the cells disintegrated over 30 min, followed by centrifugation at 4˚C and 13,500 rpm for 20 min. Soluble proteins were separated on an 8% SDS-PAGE gel, and then transferred to a nitrocellulose (NC) membrane (Whatman, Maidstone, UK) by electroblotting for 3 h at 250 mA. The membrane was blocked for 1 h at room temperature in TBS-T buffer (20 mM TRis-HCl pH 7.4, 150 mM NaCl, 0.05% Tween-20) containing 5% nonfat dry milk and washed with the buffer. The membrane was incubated with the primary antibody (1:1,000 dilution; ABcam, Cambridge, UK, ab15323) for iNOS at 4˚C overnight, washed five times, and then incubated for another 2 h with HRP-conjugated anti-rabbit IgG as a secondary antibody. The internal control was βactin antibody (1:1,000 dilution; Santa Cruz, CA, USA, sc-47778). After washing the membrane five times, the protein signals were visualized with SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Fisher, Rochford, USA) and the signal intensity measured by densitometry using Image J Software (Version 1.52a).

Antibiotics resistance tests
An Epsilometer test (E-test) was performed for quantitative minimal inhibition concentration (MIC) determination following manufacturer's protocol (bioMérieux, Stockholm, Sweden). Briefly, freeze-dried spores were diluted to 1 x 10 8 cfu/mL in distilled water. A sterile cotton swab was dipped into the inoculum, excess liquid removed, and then streaked over the entire surface of an agar plate. The plate was left for about 5 min to allow extra moisture to be absorbed by the agar. An E-test strip was then placed on the agar surface using forceps and the covered plates incubated at 37˚C for 13 to 15 h. After incubation, an inhibition zone of bacterial growth formed as an ellipse that intersected the MIC reading scale on the μg/mL unit [30].

Initial screening of strains with abundant surfactant secretion
A large number of heat-tolerant bacteria from a personal collection of insect feces and soils from remote mountains were screened to discover novel strains of surfactant-producing Bacillus species. A fine mist of oil droplets spread evenly around most of the colonies on the agar surface in the plate without a noticeable pattern (Fig 1A). However, a clearly visible halo appeared around some colonies, including a control strain of B. subtilis, ATCC21332 (hereafter ATCC21332) that abundantly secrets surfactin [9]. We tested~2,000 colonies over three months. Among the~2,000 colonies, we found 18 putatively new strains that formed halos similar to or wider than ATCC21322 (Fig 1B). The entire process was estimated to be 10 to 50 times faster than the drop-collapsing assay, so could be developed into a high-throughput screening method. The manual transfer of colonies from the initial plates to clean plates was the most time-consuming and laborious, but could be automated.

Surfactant-secreting bacteria
We chose 20 strains for identification to species level. They included the 18 strains that formed a wide, clear halo around their colony and two that did not. The 16S rRNA sequences were identical among the 18 strains with the clear zone but differed from the two strains without the zone. The 18 strains belonged to B. subtilis, the other two strains to B. cereus. For further study, we selected two of the 18 strains, ps4100 and ps4060, which respectively produced clear zones larger than or similar to ATCC21322. Although both strains abundantly secreted surfactant, the differences in the size of clear zones and growth patterns on agar plates implied that they were different strains. The size of the clear zone, not the colony, was similar but became slightly larger around ps4060 than ps4100 when they grew over a four-day period at 30˚C. In contrast, the ps4060 colony was flat and about two times wider in diameter than the convoluted colony of ps4010.

Genome features and orthologous gene phylogeny
We determined the whole genome sequences for both strains and compared them to the genomes of six other Bacillus species. The phylogeny of 16S rRNA genes indicated both strains were B. subtilis (Fig 2A). Genome size and guanine-cytosine percentage (GC%) were more similar to B. subtilis than to other Bacillus species (Table 1). Further, the numbers of rRNA operons and tRNAs were consistently identical to B. subtilis and differed from other Bacillus species, implying that both strains were B. subtilis. We inquired if these strains were identical to any of the 430 B. subtilis strains whose genome sequences were available in GenBank. A phylogeny with 1,162 orthologous genes separated ps4100 and ps4060 into two distinct groups. Strain ps4100 was a close relative of B. subtilis SRCM103882 and ps4060 belonged to a group with the B. subtilis BAB-1 strain S2 Fig in S1 File). To further investigate their phylogenetic relationship, we built a phylogenetic tree with 780 orthologous genes in 12 selected taxa and performed bootstrap analyses (Fig 2B). The strains ps4100 and ps4060 were clearly separated into two distinct groups with strong bootstrap support (Fig 2C). Bacillus subtilis SRCM103882 was 26,864 nucleotides (nt) longer and contained 47 more protein-coding sequences (CDS) than ps4100 ( Table 2), implying that they were different strains. Another outstanding difference between the two strains was a deleted region in the genome of SRCM103862 (Fig 3A). Conversely, B. subtilis BSD-2 and ps4060 differed in genome size by only 29 nt but the number of CDSs by 15 (Table 2). Although their genome organization was similar among all nine strains in this group there were spurious small-size indels across the genomes (Fig 3). In conclusion, neither strain was identical to any strain listed in GenBank.

Surfactin secretion
We compared the amount of surfactin secreted by the selected strains, ps4100 and ps4060, to ATCC21322 that produces about 0.1 mg/mL on complex media such as LB or nutrient broth [31]. Under the test conditions in this study, surfactin yields were respectively 0.291 and 0.177 mg/mL for strains ps4100 and ps4060, which was more than the 0.143 mg/mL yield of ATCC21322 under the same conditions (Fig 4A). A clear zone on the agar plate was larger around ps4060 than ps4100, probably because the ps4060 colony was two-times larger than ps4100 colony. Surfactin has at least four isoforms, differing in the length of their lipid tail [32]. HPLC profiles of surfactin secreted by ps4100 and ps4060 were similar to the standard surfactin (Sigma-Aldrich, Saint Louis, MO), with little variation in relative amounts among isoforms a, b, c, and d. ATCC21322 can secrete >3 mg/mL of surfactin under optimum conditions for surfactin production in chemically defined media [9]. Secretion capacity, however, was not compared among the three strains under these optimum conditions.

Surfactin enhances cellulase activity
We investigated the effects of surfactin on cellulase activity, which is probably important for the digestion of plant-based diets. Cellulase releases glucose from cellulose and several surfactants enhance the enzyme's activity [33]. In this study, cellulase activity decreased up to 10% in the presence of 10% methanol or Tris-HCl buffer in the reaction (Fig 4B-4D). Surfactin overcame the deleterious effects of both methanol and Tris-HCl, enhancing cellulase activity by about 20% (Fig 4B and 4C). Surfactin in water without methanol enhanced enzyme activity by up to 50% (Fig 4D). Overall, the presence of surfactin in the reaction mixture resulted in significantly more glucose released from cellulose.

Surfactin suppressed inducible NO synthetase and NO production in vitro
Pathogen infection and stresses such as weaning and sudden weather changes can induce inflammation and slow the growth of mammals [1,34,35]. We tested whether surfactin suppressed inflammation in vitro, possibly enhancing growth rates. We used four major isoforms of surfactin a, b, c, and d fractionated (S1 Fig in S1 File). None of the four major isoforms affected cell viability up to a 50 μM concentration (Fig 5A). Each isoform, however, inhibited nitric oxide (NO) production in macrophage RAW 264.7 cells activated by lipopolysaccharides (LPS) in a concentration-dependent manner. The IC 50 concentration of isoforms a, b, c, and d were 17.7, 19.5, 20.5, and 20.4 μM, respectively (Fig 5B). The inhibitory effect was strong for each isoform with little difference among them. To determine the cause of decreased NO, we analyzed the effect of each surfactin isoform on the expression of inducible NO synthetase (iNOS). This enzyme is needed to produce NO, an intracellular inflammatory mediator [36]. Ten and 25 μM of each derivative respectively caused an 80 and 90% decrease in the expression of inducible NO synthetase (iNOS, Fig 5C and 5D). The iNOS expression pattern implied that surfactin suppressed iNOS, followed by a decrease in the NO production that mediates inflammation in vivo.

Absence of antibiotic resistance
There were no antibiotic-resistant genes in the genome of either strain. Accordingly, both strains were highly susceptible to all six antibiotics tested in this study (Fig 6). Thus, these strains are suitable probiotic candidates according to the regulation set by the Korean Food and Drug Administration, a guideline concerning the assessment of bacterial susceptibility to antimicrobials of human and veterinary importance [37].

Discussion
Bacillus subtilis is generally recognized as a safe organism [38]. It is included in traditionally fermented foods, such as Korean Cheonggukjang and Japanese Natto that humans have consumed for hundreds of years. It is also safe for animal consumption. The secretion of surfactant was used in our study as a biomarker to further screen B. subtilis for potent probiotic candidates. Biosurfactant-producing bacteria can be screened using several assays, such as drop collapse, emulsification, tensinometric evaluation, fluorescence detection, and the oil- misting method [10,39,40]. We chose the oil-misting method to assess B. subtilis strains, as it was suitable for a high-throughput screening of surfactant-producing bacterial strains. After manually screening~2,000 colonies, we selected two strains, ps4100 and ps4060, mainly because they secreted more surfactin than B. subtilis ATCC21332 (Fig 1). They were novel strains (Figs 2 and 3), susceptible to antibiotics (Fig 6), thus eliminating concerns over transferring antibiotic resistance from these strains and suitable as probiotic candidates. Bacillus spp. are potentially good probiotics because they form spores that survive, germinate, and temporarily persist as vegetative cells in gastrointestinal tracts [3,4,41]. Although several strains have been developed as animal growth promoters, ps4100 and ps4060 abundantly secret surfactin and could be excellent probiotics for several reasons. The structure and amphiphilic nature of surfactin could disrupt cell membranes and inactivate or kill various microorganisms, including bacteria, viruses, fungi, and mycoplasma [42]. Although the antimicrobial efficacy of surfactin alone may not be strong, it acts synergistically with other surfactants and antimicrobial agents [43,44]. If the new Bacillus strains secret other antimicrobial bacteriocins, their efficacy would also be enhanced by surfactin [43]. Its antifungal activities could protect animals by suppressing Aspergillus and Fusarium, which produce potent mycotoxins such as aflatoxin, trichothecenes, and zearalenone [43,45]. Up to 90% of oral-fed Bacillus spores germinate and multiply as vegetative cells in the gastrointestinal tract of pigs [4]. Like antibiotics, expression of surfactin in the gastrointestinal tract would suppress the growth of disease-causing microbes and acts as animal growth promoters at a low concentration. It is notable that ps4100 and ps4060 produced about twice the surfactin as strain ATCC21332, which produces~100 μg/mL in a nutrient-rich environment and up to 600 mg/mL under optimum condition [9]. The strain ATCC21332 arguably secretes the highest amount of surfactin among known B. subtilis. The gastrointestinal tract, however, provides suboptimal conditions for Bacillus to secrete surfactin due to its high-nutrient and low-oxygen levels. Thus, the ability of ps4060 and ps4100 to secrete a large amount surfactin in a nutrient-rich environment would be an important trait against pathogenic microorganisms. There was additional evidence that surfactin-secreting Bacillus spp. might be better growth promoters than antibiotics. In our study, surfactin increased cellulase activity up to 50% in vitro (Fig 4). Cellulose is the primary component of plant cell walls, the most abundant organic molecule on earth, and the least expensive in animal diets. Efficient digestion could benefit substantially because glucose released from cellulose could reduce the use of corn and wheat as sources of starch in animal diets. It is unclear whether a 50% increase in cellulase activity is sufficient to produce a tangible effect on diet efficiency. Cellulases are generally one-to-two orders less reactive than other enzymes, such as amylases and proteases [46]. The effects of surfactin on cellulases could be substantial if the quantity of cellulase or affinity for its substrate is over ten-times higher than other enzymes. It is worth investigating if surfactin enhances the activities of other nutrient-digesting enzymes like the hemicellulases and pectin-digesting enzymes.
Surfactin may affect animal immune systems. The iNOS expression pattern in vitro implies that even at low concentrations surfactin suppresses the expression of iNOS, followed by a decrease in NO production and inflammation (Fig 5). It has a positively charged peptide loop of seven amino acids with a long hydrophobic fatty acid chain [32]. This amphiphilic structure enables it to interact with LPS that activates NO production and subsequent inflammation [47]. Lipopolysaccharide is one of two major products of gram negative bacteria among gut microbes that enter the blood stream and result in inflammation [48]. Current data are insufficient to address whether surfactin suppresses inflammation in vivo by affecting inflammationassociated genes and pathways.
In initial efficacy tests during animal growth studies, the mixture of ps4060 and ps4100 enhanced the productivity of pigs and reduced noxious gases in fecal discharges [49]. Encouragingly, novel Bacillus strains abundantly secreting surfactin in vitro appeared to be good probiotic candidates and that high-throughput screening was possible.